Stable pharmaceutical composition of tnfr:fc fusion protein

ABSTRACT

The present invention relates to the stable pharmaceutical compositions comprising tumor necrosis factor receptor Fc fusion protein (TNFR:Fc). More particularly, it relates to the stable pharmaceutical compositions comprising tumor necrosis factor receptor Fc fusion protein (TNFR:Fc), phosphate-citrate buffer. It also relates to the methods of manufacturing the composition, method of administration and kits containing the same.

This application is a National Stage Application of PCT/M2013/059612,filed 24 Oct. 2013, which claims benefit of Serial No. 1235/KOL/2012,filed 26 Oct. 2012 in India, and claims benefit of Serial No.1236/KOL/2012, filed 26 Oct. 2012 in India, and which application(s) areincorporated herein by reference. To the extent appropriate, a claim ofpriority is made to each of the above disclosed applications.

FIELD OF INVENTION

The invention provides stable pharmaceutical compositions comprisingtumor necrosis factor receptor Fc fusion protein (TNFR:Fc). Theinvention also provides methods of manufacturing the composition, methodof administration and kits containing the same.

BACKGROUND OF INVENTION

Tumor necrosis factor (TNF) alpha is a cytokine that promotes theinflammation and its associated signs by binding to its receptor. It isproduced by macrophages and many other immune cells. It is involved inpathogenesis of many inflammatory disorders like rheumatoid arthritis,psoritic arthritis, SLE, Crohn's disease etc. Hohmann et al (Hohmann etal. 1989 J Biol Chem. 25, 14927-34) identified 2 distinct receptors ofTNF-alpha which are present on different cell types viz. myeloid cellsand epithelial cells. Using monoclonal antibodies, Brockhaus et al(Brockhaus et al. 1990 ProcNatlAcadSci USA., 87(8), 3127-31)demonstrated that both TNF-alpha and beta bind to both the receptorswith high affinity.

Tumor necrosis factor-alpha (TNF-alpha) is a central regulator ofinflammation, and TNF-alpha antagonists may be effective in treatinginflammatory disorders in which TNF-alpha plays an importantpathogenetic role. Inhibition of TNF has proven to be an effectivetherapy for patients with rheumatoid arthritis and other forms ofinflammatory disease including psoriasis, psoriatic arthritis, andankylosing spondylitis, inflammatory bowel disease. One such TNF-alphaantagonist is Etanercept.

Etanercept is a dimeric fusion protein produced by recombinant DNAtechnology where gene of soluble, ligand binding portion of TNF receptor2 is fused with gene of Fc component of human IgG1 to give the desiredfusion protein (U.S. Pat. No. 7,648,702). Etanercept is expressed in CHOcells. The Fc component of Etanercept lacks CH1 domain but has CH2, CH3domains and hinge region. The fusion protein has approximate molecularweight of 150 kD and consists of 934 amino acids. Etanercept interfereswith TNF and acts as a TNF inhibitor due to which it can be used as abiopharmaceutical to treat autoimmune diseases. It prevents progressivedestruction of joints in patients with rheumatoid arthritis and thearthritis of psoriasis.

Due to its unique structure, Etanercept binds 50-100 folds moreefficiently to TNF alpha than its endogenous receptor (Gofee et. al.2003 J Am AcadDermatol. 49, S105-111, Strober 2005 SeminCutan Med Surg.24; 28-36). Additionally, due to its dimeric nature it can bind to 2 TNFalpha molecules as compared to one bound by endogenous receptor.Conjugation of this molecule to Fc region of IgG increases the half lifeas compared to endogenous soluble form. Commercially, Etanercept isavailable in both Lyophilized and liquid forms.

The most important feature of a composition is to help the protein toretain its structural conformation or its activity. The stability ofprotein in a composition can be related with long-term storage. It isunderstood to mean that the active polypeptide of the pharmaceuticalcomposition does not substantially lose its activity as compared to thecomposition at the beginning of storage.

All polypeptides have an Isoelectric Point (pI), which is generallydefined as the pH at which a polypeptide carries no net charge. It isknown in the art that protein solubility is typically lowest when the pHof the solution is equal to the isoelectric point (pI) of the protein.

The Tm of the Fab domain of a protein is a good indicator of the thermalstability of a protein and may further provide an indication of theshelf-life. Tm values of proteins determined by differential scanningcalorimetry, give insight into heat-induced changes in proteinconformation, mechanisms of protein unfolding and stabilization insolution. A lower Tm indicates less stability of a protein in givensolution, whereas a higher Tm indicates a better stability of theprotein. The Tm of the protein will vary based on the formulationcomposition which in turn reflects its stability in respectiveformulation.

During long term storage, both aqueous and lyophilized compositions ofproteins can lose active protein due to aggregation or degradation.Aggregation of the protein can lead to immunogenicity and isundesirable. Since the concentration of Etanercept used in thecomposition is high, there is a likely possibility of proteinaggregation during long term storage. To improve the stability of theprotein either the concentration of the existing excipients can bevaried or new excipients can be added to modify the composition.

U.S. Pat. No. 5,215,743; U.S. Pat. No. 7,648,702; US application US20070053906 and WO 2011141926 disclose pharmaceutical compositionscomprising aqueous composition of TNF-binding protein comprising aTNF-binding protein, a buffer and an isotonicity agent.

SUMMARY OF THE INVENTION

In an embodiment, the invention is related to a stable pharmaceuticalcomposition comprising TNFR:Fc fusion protein and phosphate-citratebuffer.

In another embodiment, the invention is related to a stablepharmaceutical composition comprising TNFR:Fc fusion protein,phosphate-citrate buffer and anti-aggregating agent selected fromL-glycine, urea and 2-hydroxypropyl beta-cyclodextrin (HPBCD).

In another embodiment, the invention is related to a stablepharmaceutical composition comprising TNFR:Fc fusion protein,phosphate-citrate buffer, anti-aggregating agent selected fromL-glycine, urea and HPBCD, a tonicity modifying agent and a stabilizingagent.

In yet another embodiment, the invention is related to the method oftreating a disease using the stable pharmaceutical composition of thepresent invention. The disease may be rheumatoid arthritis,polyarticular juvenile idiopathic arthritis, psoriatic arthritis,ankylosing spondylitis or plaque psoriasis.

In another embodiment the invention is related to a kit or containercontaining the pharmaceutical composition of the invention.

The details of one or more embodiments of the invention set forth beloware illustrative only and not intended to limit to the scope of theinvention. Other features, objects and advantages of the inventions willbe apparent from the description and claims.

DETAIL DESCRIPTION OF INVENTION

The invention provides a stable pharmaceutical composition comprisingTNFR molecules fused to an Fc portion of a human immunoglobulin (TNFR:Fcfusion protein). More particularly, the invention relates to the stablepharmaceutical composition of etanercept in phosphate-citrate buffer,which displays a lower degradation potential.

In an embodiment of the invention, the TNFR:Fc fusion protein isetanercept.

It has been reported in U.S. Pat. No. 7,648,702 patent and WO2011141926application that the pharmaceutical compositions of Etanercept usingL-glycine as anti-aggregating agent in phosphate buffer are not stableas compared to the compositions of etanercept with other amino acid suchas arginine, proline, lysine, aspartic acid as anti-aggregating agent inphosphate buffer. The WO2011141926 application discloses that thecomposition comprising Etanercept in phosphate buffer and L-glycine asanti-aggregating agent showed aggregation as well as fragmentationproducts.

While studying the Etanercept compositions in different buffers andusing different anti-aggregating agents it was observed that Etanerceptcomposition comprising phosphate-citrate buffer with L-glycine asanti-aggregating agent showed improved stability as compared toEtanercept composition comprising phosphate buffer with L-glycine asanti-aggregating agent at 5° C. and 40° C.

As illustrated in the example section, the stability of Etanerceptcomposition essentially consisting of phosphate-citrate buffer incombination with L-glycine as anti-aggregating agent were assessedduring a 6 months stability study at 5° C. as well as 2 weeks study at40° C. (stress conditions stability studies). Compositions comprisingthe phosphate-citrate buffer system were determined to be superior ascompared to composition comprising phosphate buffer system with respectto the % aggregation products and & degradation products as determinedby SEC.

The stable pharmaceutical composition used herein means that the TNFR:Fcfusion protein exhibits following features:

-   -   i. The stable pharmaceutical composition of TNFR:Fc fusion        protein in phosphate-citrate buffer exhibits improved stability        as compared to the composition of etanercept comprising        phosphate buffer, arginine and sodium chloride. The % aggregates        are less in the Etanercept composition comprising L-glycine as        anti-aggregating agent in phosphate-citrate buffer as determined        after 2 weeks of storage at 40° C. by SEC.    -   ii. The stable pharmaceutical composition of TNFR:Fc fusion        protein in phosphate-citrate buffer exhibits less than 5% high        and low molecular weight impurities similar to the innovator        composition of etanercept comprising phosphate buffer, arginine        and sodium chloride after 2 weeks of storage at 40° C. by SEC.    -   iii. The stable pharmaceutical composition of TNFR:Fc fusion        protein in phosphate-citrate buffer, glycine exhibits        approximately 8% high and low molecular weight impurities.        Whereas the composition of etanercept comprising phosphate        buffer, glycine as an anti-aggregating agent showed ˜15%        impurities, after 2 weeks of storage at 40° C. by SEC.

In another embodiment the invention relates to the pharmaceuticalcomposition of Etanercept in phosphate-citrate buffer with otheranti-aggregating agents such as urea, HPBCD.

After obtaining improved stability of Etanercept composition inphosphate-citrate buffer other anti-aggregating agents from other classof compounds than amino acids were tested.

It was observed that Urea and HPBCD also provided stable pharmaceuticalcompositions of Etanercept in phosphate-citrate buffer. It is understoodto mean that etanercept of the pharmaceutical composition does notsubstantially lose its activity as compared to the composition at thebeginning of storage. The term ‘substantially’ refers to not more than20%, or more preferably 15%, or even more preferably 10%, and mostpreferably 5% of its activity relative to activity of the composition atthe beginning of storage. The pharmaceutical composition of theinvention is suitable for long term storage. As used herein, ‘the longterm storage’ means that the storage of the pharmaceutical compositionis stable for more than a month, preferably more than 6 months or 12months, more preferably more than 24 months.

Tumor Necrosis Factor alpha (TNF-alpha) is a member of a group ofcytokines that stimulate the acute phase reaction, and thus is acytokine involved in systemic inflammation. TNF-alpha is able to induceinflammation, induce apoptotic cell death, and to inhibit tumorgenesisand viral replication. Dysregulation of TNF-alpha production has beenimplicated in a variety of human diseases like autoimmune disease,ankylosing spondylitis, juvenile rheumatoid arthritis, psoriasis,psoriatic arthritis, rheumatoid arthritis, Wegener's disease(granulomatosis), Crohn's disease or inflammatory bowel disease, chronicobstructive pulmonary disease (COPD), Hepatitis C, endometriosis,asthma, cachexia, atopic dermatitis, Alzheimer as well as cancer.

Dosage of the TNFR:Fc will depend on the disease, severity of condition,patient's clinical history, and response to the (prior) therapy, andwill be adjusted and monitored by a physician. The pharmaceuticalcomposition may be administered parenterally, such as subcutaneously,intramuscularly, intravenously, intraperitoneally, intracerebrospinally,intra-articularly, intrasynovially and/or intrathecally by either bolusinjection or continuous infusion.

In an embodiment the TNFR:Fc may be administered in adult or juvenilesubject, wherein the amount may range from about 1-80 mg. The dose maybe administered once weekly, twice weekly. Further, the doses may beadministered weekly, biweekly, or separated by several weeks e.g. threeweeks. The therapeutic dose and duration may vary as per patientresponse and patient requirement.

In another embodiment, a suitable regimen for juvenile and pediatricpatients may involve a dose of 0.4 mg/kg to 5 mg/kg of TNFR:Fc,administered one or more times per week.

In case of adult rheumatoid arthritis, 25 mg twice weekly or 50 mg onceweekly TNFR:Fc may be administered.

In case of psoriatic arthritis, 25 mg twice weekly or 50 mg once weeklyTNFR:Fc may be administered.

In case of Ankolysing spondylitis, 25 mg twice weekly or 50 mg onceweekly TNFR:Fc may be administered.

In case of adult plaque psoriasis, the recommended dose of TNFR:Fc is 25mg administered twice weekly or 50 mg administered once weekly. In caseof pediatric plaque psoriasis, the recommended dose of TNFR:Fc is 0.8mg/Kg weekly with a maximum of 50 mg dose per week.

In case of polyarticular juvenile idiopathic arthritis, the recommendeddose of TNFR:Fc is 0.8 mg/Kg weekly with a maximum of 50 mg dose perweek.

In case of renal and hepatic impairment no dose adjustment is required.

In a second aspect, the invention relates to a kit comprising acomposition according to the first aspect and instructions for use ofthe present composition.

In a preferred embodiment, the composition is contained in a pre-filledsyringe. In another preferred embodiment, the composition is containedin a pre-filled vial. The kit may comprise one or more unit dosage formscontaining the pharmaceutical composition of the invention.

Any suitable syringe or vial or cartridge may be used. The kit may alsocomprise the pharmaceutical composition according to the invention inanother secondary container, such as in an autoinjector. The prefilledsyringe may contain the composition in aqueous form. Described syringemay be further supplied with an autoinjector, which often is adisposable article for single use only, and may e.g. have a volumebetween 0.1 and 1 ml. However, the syringe or autoinjector may also befor multi-usage or multi-dosing. The described vial may contain thecomposition in lyophilised or aqueous state, and may serve as a singleor multiple use device. The vial may e.g. have a volume between 1 and 10ml.

The pharmaceutical composition is sterile and stable for long period oftime at 2-8° C. Also it is stable up to 6 months when stored at 25° C.The invention provides pharmaceutical composition essentially comprisingof etanercept, phosphate-citrate buffer, anti-aggregating agent selectedfrom L-glycine, urea and HPBCD, a tonicity modifier, a stabilizer andoptionally other excipients in suitable combination thereof.

The invention further relates to a stable pharmaceutical composition,wherein the composition is liquid or lyophilized. The invention isfurther related to a stable pharmaceutical composition in a pre-filledsyringe, vial, cartridge, or pen.

In an embodiment of the invention, the active pharmaceutical ingredientetanercept is used which is obtained from recombinant DNA technologyusing CHO cells. The concentration of the etanercept in the compositionis 10 mg/mL to 100 mg/mL. In a preferred embodiment of the invention,the concentration of etanercept in the composition is 10 mg/mL to 60mg/mL. In the most preferred embodiment of the invention, theconcentration of etanercept in the composition is 20 mg/mL to 60 mg/mL.

In another embodiment of the invention, the buffer is phosphate-citratebuffer. In an embodiment of the invention, the concentration of thebuffer in the composition is 10 mM to 100 mM. In a preferred embodimentof the invention, the concentration of the buffer in the composition is10 mM to 50 mM. In another preferred embodiment of the invention, theconcentration of the buffer in the composition is 20 mM to 40 mM.

In another embodiment of the invention, the pH of the composition is 5to 8. In another embodiment of the invention, the etanercept compositioncomprises anti-aggregating agent selected from L-glycine, urea andHPBCD.

In an embodiment of the invention when the anti-aggregating agent isL-glycine, then the concentration of L-glycine in the composition is 10mM to 300 mM.

In another embodiment of the invention when the anti-aggregating agentis urea, the concentration of urea in the composition is 20 mM to 50 mM.

In another embodiment of the invention when the anti-aggregating agentis HPBCD, then the concentration of HPBCD is 10 mM to 100 mM.

In another embodiment of the invention, the stable pharmaceuticalcomposition further comprises a parenterally acceptable tonicity agent.The tonicity agent is selected from the group of salts such as sodiumchloride, potassium chloride, calcium chloride or saccharides such asmannitol, sucrose, glucose, or amino acids such as arginine, cysteine,histidine and the like. The preferred tonicity agent is sodium chloride.The concentration range varies from 0 mM to 150 mM.

In yet another embodiment of the invention, the stable pharmaceuticalcomposition further comprises a stabilizer. The stabilizer is selectedfrom the group consisting of sucrose, trehalose, lactose, mannitol. Thepreferred stabilizing agent is sucrose. The concentration of thestabilizing agent in the composition varies from 0.5 wt % to 10 wt %. Inthe most preferred embodiment of the invention, the concentration of thestabilizer in the composition is 0.5 wt % to 1.5 wt %.

In yet another embodiment of the invention, the stable pharmaceuticalcomposition may optionally comprise a chelating agent. The chelatingagent is selected from the group consisting of EDTA, DTPA, HEDTA, NTAand TSP. The preferred chelating agent is EDTA. In a more preferredembodiment of the invention, the concentration of EDTA is 0 mM to 10 mM.In another embodiment of the invention, the stable pharmaceuticalcomposition of the invention comprises stable etanercept,phosphate-citrate buffer; anti-aggregating agent selected fromL-glycine, urea or HPBCD; sucrose as a stabilizing agent and with a longshelf life at temperature 5° C.

In another embodiment of the invention, the stable pharmaceuticalcomposition of the invention comprises stable etanercept,phosphate-citrate buffer; anti-aggregating agent selected fromL-glycine, urea or HPBCD; sucrose as a stabilizing agent and with a longshelf life at 5° C.

In another embodiment of the invention, the stable pharmaceuticalcomposition of the invention comprises stable etanercept,phosphate-citrate buffer; anti-aggregating agent selected fromL-glycine, urea or HPBCD; sucrose as a stabilizing agent and with 2weeks shelf life at 40° C.

In another embodiment of the invention, the stable pharmaceuticalcomposition of the invention comprises stable etanercept,phosphate-citrate buffer; anti-aggregating agent selected fromL-glycine, urea or HPBCD; sucrose as a stabilizing agent which providesbetter stability to the pharmaceutical composition to maintain itsactivity for the longer period of time providing longer shelf life.

In another embodiment, the invention pertains to a method of producing apharmaceutical composition according to the first aspect, comprisingTNFR:Fc, phosphate-citrate buffer, stabilizing agent selected from thegroup consisting of L-glycine, urea and HPBCD.

In a preferred embodiment, the method may further comprise the step ofadding at least one tonicity modifier, such as sodium chloride; astabilizer, such as sucrose and optionally a chelating agent as definedabove.

In another embodiment, the method may further comprise a lyophilizationstep, which may be before or after adding the at least one tonicitymodifier, and/or an excipient as defined above.

Accordingly to a preferred embodiment of the present invention thepharmaceutical composition comprises 10 mg/ml to 100 mg/ml ofetanercept, about 10 mM to 100 mM of phosphate citrate buffer, about 10mM to 300 mM L-glycine, about 0 mM to 150 mM sodium chloride and about0.5 wt % to 10 wt % sucrose having a pH range of 5 to 7.

Accordingly to another preferred embodiment of the present invention thepharmaceutical composition comprises 10 mg/ml to 100 mg/ml ofetanercept, about 10 mM to 100 mM of phosphate citrate buffer, about 1mg/ml to 18 mg/ml urea, about 1 mM to 150 mM sodium chloride, about 0.5wt % to 2 wt % sucrose and about 0 mM to 10 mM EDTA having a pH range of5 to 7.

In another preferred embodiment of the present invention thepharmaceutical composition comprises 10 mg/ml to 100 mg/ml ofetanercept, about 10 mM to 100 mM of phosphate citrate buffer, about 20mg/ml to 30 mg/ml HPBCD, about 1 mM to 150 mM sodium chloride, about 0.5wt % to 2 wt % sucrose and about 0 mM to 10 mM EDTA having a pH range of5 to 7.

The invention will be more fully understood by reference to thefollowing examples. However, the examples should not be construed aslimiting the scope of the invention.

EXPERIMENTAL SECTION

The active ingredient etanercept, which was used for the describedexamples, is derived from recombinant DNA technology in CHO cells. TheCHO cells were cultured in a fed-batch process. Etanercept was purifiedfrom the cell free harvest by standard purification and filtrationprocess including affinity chromatography and further chromatographicand filtration steps. Etanercept was derived from different productionbatches which was used in the examples, i.e., example 2 and example 5.

General Process for Preparation of Stable Pharmaceutical Composition ofEtanercept

The process for preparing the Etanercept drug substance compositionscomprises of 2 steps viz. preparation of formulated bulk and fillfinish. The formulated bulk is prepared by diluting the drug substancewith the formulation buffer to achieve the desired concentration of drugproduct. The formulation buffer is prepared by adding required quantityof Trisodium Citrate dihydrate and Sodium dihydrogen phosphate dihydrateto WFI followed by mixing. Further, required quantities of otherexcipients are added to the above solution and the desired volume isadjusted with WFI after adjustment of pH. The formulation buffer is thenaseptically filtered using 0.22μ sterilizing grade PVDF filter. As perthe batch calculation, the required quantity of the Etanercept (in sameformulation) is aseptically diluted

The compositions were analysed by Size Exclusion-High-performance liquidchromatography (SE-HPCL) at different time frames of storage at 5° C.,25° C. and 40° C. SE-HPLC separates the proteins and its relatedimpurities on the basis of their size. Therefore, it is useful to detectaggregation and fragmentation of Etanercept.

The examples which follow are illustrative of the invention and are notintended to be limiting.

Example 1

The process for preparing the Etanercept drug substance compositions wascomprises of 2 steps viz. preparation of formulated bulk and fillfinish. The formulated bulk is prepared by diluting the drug substancewith the formulation buffer to achieve the desired concentration of drugproduct. The formulation buffer is prepared by adding required quantity(as mentioned in table 1) of Trisodium Citrate dihydrate and Sodiumdihydrogen phosphate dihydrate to WFI followed by mixing. Further,required quantities of Glycine as anti-aggregating agent and otherexcipients are added to the above solution and the desired volume isadjusted with WFI after adjustment of pH. The formulation buffer is thenaseptically filtered using 0.22μ sterilizing grade PVDF filter. As perthe batch calculation, the required quantity of the Etanercept DS (insame formulation) is aseptically diluted with the filtered formulationbuffer to achieve the desired concentration of 50±5 mg/mL of Etanerceptbulk. The formulated bulk is filtered through 0.22μ sterilizing gradePVDF filter and is aseptically dispensed into prefilled syringes. ThePFSs were then charged on stability at various temperatures. Table 1describes the composition obtained using L-glycine as anti-aggregatingagent in 75 mM concentration.

TABLE 1 The composition of Example 1 Excipients Concentration MolarConcentration Etanercept  50 mg/mL 50 mg/mL NaH2PO4 dihydrate 2.6 mg/mL16.6 mM Trisodium Citrate 4.5 mg/mL 15.3 mM dihydrate Sucrose  10 mg/mL1% NaCl 3.8 mg/mL   65 mM L-glycine 5.6 mg/mL   75 mM

Example 2

Etanercept compositions were studied in different buffers and usingdifferent anti-aggregating agents. The short term stability ofEtanercept composition comprising phosphate-citrate buffer withL-glycine as anti-aggregating agent was analysed at 5° C. for 6 monthsand 40° C. for 2 weeks along with the Etanercept composition comprisingphosphate buffer with L-glycine as anti-aggregating agent, where otherexcipients were maintained constant by using SE-HPLC and the results areprovided in Table 2. The Table 2 illustrates the stability studies ofdifferent etanercept compositions.

TABLE 2 Stability studies at 5° C. and 40° C. Etanercept in Etanerceptin phosphate citrate buffer phosphate buffer with L-glycine withL-glycine % purity on day 1 92.7 91.2 @ 5° C. % purity after 6 months85.6 68.2 @ 5° C. % purity on day 1 90.9 89.3 @ 40° C. % purity after 2weeks 82.8 74.9 @ 40° C.

As table 2 illustrates, Etanercept composition comprisingphosphate-citrate buffer with L-glycine as anti-aggregating agent showedimproved stability as compared to Etanercept composition comprisingphosphate buffer with L-glycine as anti-aggregating agent at 5° C. and40° C.

Example 3

The process for preparing the Etanercept drug substance composition issimilar as explained in example 1, wherein the anti-aggregating used isurea. Composition shown in Table 3 was prepared using urea asanti-aggregating agent.

TABLE 3 The composition of Example 3 Excipients Concentration MolarConcentration Etanercept  50 mg/mL 50 mg/mL NaH2PO4 2.6 mg/mL 16.6 mMTrisodium Citrate 4.5 mg/mL 15.3 mM Sucrose  10 mg/mL 1% NaCl 5.8 mg/mL 100 mM EDTA 1.8 mg/mL   5 mM Urea 1.5 mg/mL   25 mM

Example 4

The process for preparing the Etanercept drug substance composition issimilar as explained in example 1, wherein the anti-aggregating used isHPBCD. Composition shown in Table 4 was prepared using HPBCD asanti-aggregating agent.

TABLE 4 The composition of Example 4 Excipients Concentration MolarConcentration Etanercept  50 mg/mL 50 mg/mL NaH2PO4 2.6 mg/mL 16.6 mMTrisodium Citrate 4.5 mg/mL 15.3 mM Sucrose  10 mg/mL 1% NaCl 5.8 mg/mL 100 mM EDTA 1.8 mg/mL   5 mM HPBCD  25 mg/mL 17.8 mM

Example 5

The innovator composition of etanercept comprising phosphate buffer andarginine as anti-aggregating agent (Composition 1) and the compositionof etanercept comprising phosphate-citrate buffer and L-glycine asanti-aggregating agent (Composition 2) were filled in PFSs and werecharged on long term stability which is ongoing. The data of proteinpurity after 9 months storage at 5° C., 6 months storage at 25° C., 2weeks storage at 40° C. was analysed by using SE-HPCL and the resultsare provided in table 5.

TABLE 5 Comparative stability data Composition Composition 1 2 % purityon day 0 97.5 97.6 % purity after 9 months @ 5° C. 96.9 96.9 % purityafter 6 months @ 25° C. 94.1 93.5 % purity after 2 weeks @ 40° C. 95.395.1

Similarly, the compositions of example 3 and example 4, i.e.,composition of etanercept comprising phosphate-citrate buffer with ureaas anti-aggregating agent and composition of etanercept comprisingphosphate-citrate buffer with HPBCD as anti-aggregating agent werefilled in PFSs and were studied for stability at 5°, 25° and 40° C. Thestability of these compositions was assessed and was found comparablewith the composition 1 up to period of 2 weeks.

Example 6

Etanercept used for the lyophilization studies is formulated anddialyzed extensively with pharmaceutical compositions mentioned in table6. Respective formulated bulks are filled in vials, half stoppered andare subjected to lyophilization.

TABLE 6 The compositions of Example 6 Excipients Composition 3Composition 4 Etanercept   50 mg/mL   50 mg/mL NaH2PO4 dihydrate  2.6mg/mL  2.6 mg/mL Trisodium Citrate  4.5 mg/mL  4.5 mg/mL dihydrateSucrose    3 mg/mL    1 mg/mL L-glycine 11.24 mg/mL 22.48 mg/mL

All patents, patent applications and publications cited in thisapplication are hereby incorporated by reference in their entirety forall purposes to the same extent as if each individual patent, patentapplication or publication were so individually denoted.

Although certain embodiments and examples have been described in detailabove, those having ordinary skill in the art will clearly understandthat many modifications are possible in the embodiments and exampleswithout departing from the teachings thereof.

1.-20. (canceled)
 21. A stable liquid pharmaceutical composition comprising Etanercept phosphate-citrate buffer, glycine, sucrose, and sodium chloride.
 22. The pharmaceutical composition of claim 21 comprising 10 mg/ml to 100 mg/ml of Etanercept, about 10 mM to 100 mM of phosphate-citrate buffer, about 10 mM to 300 mM glycine, about 50 mM to 120 mM sodium chloride and about 0.5 wt % to 10 wt % sucrose.
 23. The stable pharmaceutical composition of claim 21 wherein the composition has a pH in the range of 5 to
 7. 24. The stable pharmaceutical composition of claim 21 wherein the composition is sterile and ready for parenteral administration.
 25. The pharmaceutical composition of claim 21 comprising 10 mg/ml to 100 mg/ml of Etanercept, about 10 mM to 100 mM of phosphate-citrate buffer, about 10 mM to 300 mM glycine, about 0 mM to 150 mM sodium chloride and about 0.5 wt % to 10 wt % sucrose.
 26. The pharmaceutical composition of claim 22 comprising 50 mg/ml of Etanercept about 16.6 mM of phosphate buffer, about 15.3 mM of citrate buffer, about 75 mM glycine, about 65 mM sodium chloride and about 1 wt % sucrose.
 27. The pharmaceutical composition of claim 22 comprising 50 mg/ml of Etanercept about 31.9 mM of phosphate-citrate buffer, about 75 mM glycine, about 65 mM sodium chloride and about 1 wt % sucrose. 